There exists a fundamental gap in our understanding of the relationship between pyranopterin molybdenum enzyme structure and function. The long-term goals of our research are to understand enzyme mechanism in order to improve the quality of human health and the environment. Our objective in pursuit of these goals is to develop a comprehensive understanding of how active site geometric and electronic structure contributes to proper enzyme function. This will be accomplished through a combination of detailed spectroscopic (electronic absorption, MCD, Raman, XAS, EPR, etc.) and bonding studies on enzymes from all three pyranopterin Mo enzyme families. This work will be complemented by parallel studies on small molecule analogues. Our combined spectroscopic approach is designed to provide detailed insight into key electronic structure contributions to catalysis and our computational studies will be calibrated to spectroscopic and reactivity data in order to obtain a high level of mechanistic detail. The central hypothesis is that a complex interplay exists between active site geometric and electronic structure that functions to facilitate the unique reactions these enzymes catalyze. The rationale for this research is that a comprehensive understanding of how electronic structure contributes to reactivity in pyranopterin Mo enzymes will lead to greater insight into innovative drug and pro-drug design, understanding disease states related to Mo enzyme activity, and generally improving human health and the environment. We will test our central hypothesis in order to accomplish the stated objective of this proposal through the successful pursuit of the three Specific Aims 1) Determine the reaction coordinate for the molybdenum hydroxylases, 2) Develop a comprehensive understanding of active site contributions to catalysis in the sulfite oxidase family enzymes YedY and mARC, and 3) Identify key molybdenum-sulfur covalency contributions to electron transfer (ET) and redox potential modulation in dimethylsulfoxide reductase family enzymes. Our research plan is innovative because it 1) utilizes a combined spectroscopic approach coupled with sophisticated computational studies to probe key enzyme states with minimal or no interference from endogenous chromophores, 2) proposes to study a new Mo enzyme found in humans (mARC), and 3) contributes to a greater understanding of the pyranopterin dithiolene in catalysis. This proposed research is significant because it will lead to a markedly greater understanding of how active site geometric and electronic structure directly affect molybdoenzyme substrate specificity and the nature of the reaction coordinate, the nature of the reaction catalyzed (oxidation/reduction), and the role of the pyranopterin dithiolene cofactor in catalysis.